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Improved selectivity of an engineered multi-product terpene synthase

Lauchli, Ryan and Pitzer, Julia and Kitto, Rebekah Z. and Kalbarczyk, Karolina Z. and Rabe, Kersten S. (2014) Improved selectivity of an engineered multi-product terpene synthase. Organic and Biomolecular Chemistry, 12 . pp. 4013-4020. ISSN 1477-0520.

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Mutation of the sesquiterpene synthase Cop2 was conducted with a high-throughput screen for the cyclization activity using a non-natural substrate. A mutant of Cop2 was identified that contained three amino acid substitutions. This mutant, 17H2, converted the natural substrate FPP into germacrene D-4-ol with 77% selectivity. This selectivity is in contrast to that of the parent enzyme in which germacrene D-4-ol is produced as 29% and α-cadinol is produced as 46% of the product mixture. The mutations were shown to each contribute to this selectivity, and a homology model suggested that the mutations lie near to the active site though would be unlikely to be targeted for mutation by rational methods. Kinetic comparisons show that 17H2 maintains a k_(cat)/K_M of 0.62 mM^(−1) s^(−1), which is nearly identical to that of the parent Cop2, which had a k_(cat)/K_M of 0.58 mM^(−1) s^(−1).

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Additional Information:© 2014 Royal Society of Chemistry. Received 3rd March 2014, Accepted 9th April 2014. First published online 10 Apr 2014. We are grateful to Frances H. Arnold, Claudia Schmidt- Dannert, David Cane, Scott Virgil, Robert M. Coates, and David Christianson for their pioneering studies, invaluable advice, encouragement, and materials. We thank Sabine Brinkmann-Chen, John McIntosh, Thomas Heel, and Chris Farwell for advice and assistance. RL acknowledges the support of the NIH fellowship 1F32GM095061. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. KSR thanks the Deutscher Akademischer Austauschdienst (DAAD) for a postdoctoral fellowship. KZK and RZK acknowledge the support of Summer Undergraduate Research Fellowships from the California Institute of Technology.
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NIH Fellowship1F32GM095061
Deutscher Akademischer Austauschdienst (DAAD) postdoctoral fellowshipUNSPECIFIED
Caltech Summer Undergraduate Research Fellowship (SURF)UNSPECIFIED
Record Number:CaltechAUTHORS:20140521-142650768
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Official Citation: Improved selectivity of an engineered multi-product terpene synthase Ryan Lauchli, Julia Pitzer, Rebekah Z. Kitto, Karolina Z. Kalbarczyk and Kersten S. Rabe Org. Biomol. Chem., 2014,12, 4013-4020 DOI: 10.1039/C4OB00479E, Paper
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:45874
Deposited By: Ruth Sustaita
Deposited On:27 May 2014 17:01
Last Modified:03 Oct 2019 06:37

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